We are developing HPIVs and APMVs as vaccine vectors for human use against highly pathogenic emerging viruses, using HPIV3 and NDV as proof of principle. Vectored vaccines are particularly well suited for highly pathogenic agents because they do not involve the pathogen in infectious form and thus are much safer to develop, manufacture, and use. Vectors can be evaluated proactively with test antigens, gaining valuable clinical experience that can expedite vaccine development against new pathogens in real time outbreaks. Although a number of DNA or viral vectors are under development by various laboratories worldwide, each has particular strengths and deficiencies. Also, immunization against multiple pathogens will require multiple antigenically-distinct vectors, to avoid interference by vector-specific immunity. Our vectors are unusual in that they take advantage of the natural tropism of HPIVs and NDV to provide respiratory administration and direct stimulation of mucosal immunity. The HPIV and NDV vectors have several advantages. They are relatively easy to make, grow, and manufacture as clinical trial material (we have considerable experience with the HPIVs). They are administered to the respiratory tract by nasal drops (we have done many such clinical trials with the HPIVs and related viruses), spray, or a WHO aerosol device that has been used by others in the field studies for measles virus vaccine (and that we have used in non-human primates with NDV vectors). The respiratory route stimulates strong systemic immune responses, and also stimulates local mucosal immunity that is important for restricting pathogens that infect and are spread by contact and self-inoculation (e.g., from body fluids or fomites). In primates, the replication of HPIV and NDV is restricted to the respiratory tract, and this restricted tropism reduces safety concerns compared to systemic viruses. HPIV and NDV vectors are given by one or two doses. Regarding the HPIVs, we previously showed that a single immunization of African green monkeys with a PIV3-based vector (an attenuated chimera of HPIV3 and BPIV3 called B/HPIV3) expressing the SARS spike S glycoprotein induced immunity sufficient to prevent shedding by a high-dose IN/IT challenge of SARS-CoV. We also previously showed that a single immunization of rhesus monkeys with HPIV3 expressing glycoprotein GP of Ebola virus (EV) was 78% effective in preventing mortality from an EV challenge, and two doses provided sterilizing immunity and protection in all of the animals. This past year, we further evaluated HPIV3 as a vector for glycoprotein GP of Ebola virus using the aerosol route of administration. In rhesus monkeys, a single aerosol dose provided complete protection against challenge (no detectable challenge Ebola virus replication) with an otherwise lethal dose of Ebola virus. The level of neutralizing serum antibody induced by this single dose was substantially and significantly greater than that induced by an alphavirus vector expressing Ebola GP evaluated in parallel, and was equivalent to that induced by two doses of the alphavirus vector expressing Ebola GP. We also previously created a version of HPIV3-Ebola-GP in which the HPIV3 F and HN genes were deleted, leaving Ebola GP as the sole viral surface antigen. In guinea pigs, this virus was very highly attenuated, but a single immunization induced sterilizing immunity against Ebola challenge. This virus presently is being evaluated in rhesus monkeys. A second strategy has been to investigate APMVs as vectors, in particular NDV. We previously showed that NDV is naturally restricted in non-human primates due to host range differences. Indeed, most immunized animals did not detectably shed NDV, and direct analysis of lung tissue revealed very low levels of replication. NDV-based vectors expressing protective antigens of Ebola virus, SARS, or HPAIV were immunogenic and protective (evaluated for SARS and HPAIV) against the respective pathogen in non-human primates, although two doses were used due to the high level of attenuation of the vector. We mostly have used the mesogenic (intermediate virulence) NDV strain Beaudette C as a vector. However, mesogenic NDV was classified as a Select Agent several years ago, which complicates and limits its use. Low-virulence (lentogenic) strains of NDV are not Select Agents but may be suboptimally immunogenic. Therefore, in the past few years, we have been modifying the mesogenic BC strain with the goal that it would no longer qualify as a Select Agent but would retain its effectiveness as a vaccine vector. For example, we changed the multibasic cleavage site sequence of the F protein to the dibasic sequence of lentogenic (avirulent) strain LaSota. Additionally, the BC F and HN proteins were modified in several ways to reduce virulence while maintaining or even enhancing virus replication. This included replacing the BC HN gene with that of La Sota, and replacing segments of the BC F protein with their counterparts from a velogenic (highly virulent) African AKO NDV strain. These modified BC-derived vectors and the control LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 HPAIV as a test foreign antigen. In general, the modified BC-based vectors expressing HA replicated better than the LaSota/HA control, and expressed higher levels of HA protein. Pathogenicity tests indicated that all of the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with NDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271-330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy. NDV was evaluated as a vaccine vector to express HIV-1 Env protein gp160, gp120, and two versions of gp140 that differed slightly in length (gp140S and L). NDV-expressed gp160, gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Prime-boost intransal immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein. NDV is APMV serotype 1. Other serotypes exist. We evaluated the following APMV serotypes for attenuation in rhesus monkeys, as potential vaccine vector candidates: wild type APMV-2, -3, -4, -5, -7 and -9, and versions of APMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for disease, virus shedding, and seroconversion. The results indicated that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype, and induced serum antibody responses. Modification of the F protein had little effect. None of the animals exhibited any clinical disease signs. These results suggest that these viruses represent promising vector candidates.